Summary: Many of our most effective viral vaccines are live-attenuated viruses. However, for HIV vaccines, it seems that virtually no degree of attenuation would be safe enough for human use. As a surrogate, a number of live recombinant vaccines have been proposed. These have generally beeen defective viruses, capable of expressing HIV antigens in a single round of infection, but unable to replicate further. In contrast, we have undertaken development of a live viral vector safe enough to express HIV antigens over multiple rounds of viral replication. Like HIV, rubella is an enveloped plus strand RNA virus which infects at mucosal surfaces. A single dose protects for life against mucosal and systemic infection with rubella virus. Live attenuated rubella vaccine is safe for human use. It has been given safely to millions of children around the world, including asymptomatic HIV+ children. It has no DNA intermediate, so there is no risk of integration or reservoir of chronic infection. It is minimally pathogenic for adults and children and its genetic stability has such that it has not changed serotype in over 40 years. Ideally, a rubella/HIV hybrid could combine the growth and safety of rubella with the antigenicity of HIV gp120, but without the pathogenicity of HIV. Potentially, it may be possible with this vaccine to induce a level and duration of immunity to HIV comparable to what has been achieved for rubella. We are using a full length infectious cDNA clone of rubella, provided by Dr. Teryl Frey. We are actively searching for acceptor sites on rubella, where foreign sequences can be inserted without disrupting essential viral functions. When grown in cell culture, rubella accumulates defective interfering virus, which have deletions in a structural gene but contain all essential cis-acting elements needed for second strand RNA synthesis and replication. Our strategy is to use the sites of these deletions to insert a GFP reporter gene into full length rubella cDNA, while keeping all viral genes intact. These constructs will be transcribed to infectious RNA and transformed into VERO cells in the presence or absence of helper virus. Growth of the rubella/GFP hybrid will be detected as green fluorescence, as well as cytopathic effects. Once we have identified an insertion site, we will insert a gp120 gene there to produce an infectious rubella/gp120 hybrid. The hybrid virus will be tested for growth and gp120 expression in vitro. It will then be tested for propagation in rhesus macaques and for the ability to elicit anti-gp120 antibodies (serum IgG and mucosal IgA), as well as safety and viral shedding and persistence. Macaques are the ideal host for demonstrating protection, since they can be infected with rubella and then challenged with an SHIV or SIV challenge strain expressing envelope glycoproteins of the same type or different from the vaccine strain.